Application of surface plasmon resonance technology for detecting and genotyping hpv

ABSTRACT

The present invention discloses using SPR technology to simultaneously and qualitatively detect different HPV genotypes. It also discloses an efficient formula to make a mixed SAM that can greatly enhance the immobilization ability of the metal surface in SPR based techniques, which is good for the immobilization of HPV specific DNA probes used for the detection of different HPV genotypes.

TECHNICAL FIELD

The present invention relates to a method of using SPR technology tosimultaneously detect HPV genotypes for the diagnosis of HPV infection.

INDUSTRIAL APPLICABILITY

It has been recognized that it would be advantageous to advantageous todevelop a label-free and high-throughput technique to detect HPVgenotypes. SPR technology has the characteristics of providingunlabeled, high-throughput, and on-line parallel analysis. In thisinvention, we employed SPR technology to detect HPV genotypes for thediagnosis of HPV infection. Briefly, and in general terms, the inventionis directed to the application of SPR technology in detecting HPVgenotypes, which is significant for the diagnosis of HPV infection.

DISCLOSURE OF THE INVENTION

Surface plasmon resonance (SPR) technology has been employed forquantitative and qualitative analysis in analytical chemistry,biochemistry, physics and engineering. SPR technology has become aleading technology in the field of direct real-time observation ofbiomolecular interactions.

SPR technology is highly sensitive to changes that occur at theinterface between a metal and a dielectric medium (e.g., water, air,etc). In general, a high-throughput SPR instrument consists of anauto-sampling robot, a high resolution CCD (charge-coupled device)camera, and gold or silver-coated glass slide chips each with more than4 array cells embedded in a plastic support platform.

SPR technology exploits surface plasmons (special electromagnetic waves)that can be excited at certain metal interfaces, most notably silver andgold. When incident light is coupled with the metal interface at anglesgreater than the critical angle, the reflected light exhibits a sharpattenuation (SPR minimum) in reflectivity owing to the resonant transferof energy from the incident light to a surface plasmon. The incidentangle (or wavelength) at which the resonance occurs is highly dependentupon the refractive index in the immediate vicinity of the metalsurface. Binding of biomolecules at the surface changes the localrefractive index and results in a shift of the SPR minimum. Bymonitoring changes in the SPR signal, it is possible to measure bindingactivities at the surface in real time. Traditional SPR spectroscopysensors, which measure the entire SPR curve as a function of angle orwavelength, have been widely used, but offer limited throughput. Thehigh-throughput capability of a high-throughput SPR instrument islargely due to its imaging system. The development of SPR imaging allowsfor the simultaneous measurement of thousands of biomoleculeinteractions.

Typically, a SPR imaging apparatus consists of a coherent p-polarizedlight source expanded with a beam expander and consequently reflectedfrom a SPR active medium to a detector. A CCD camera collects thereflected light intensity in an image. SPR imaging measurements areperformed at a fixed angle of incidence that falls within a linearregion of the SPR dip; changes in light intensity are proportional tothe changes in the refractive index caused by binding of biomolecules tothe surface. As a result, gray-level intensity correlates with theamount of material bound to the sensing region. In addition, one of thefactors determining the sensitivity of a SPR imaging system is theintensity of the light source. The signal strength from the metalsurface is linearly proportional to the incoming light strength, so alaser light source is preferred over light-emitting diode and halogenlamps.

The SPR instrument is an optical biosensor that measures binding eventsof biomolecules at a metal surface by detecting changes in the localrefractive index. The depth probed at the metal-aqueous interface istypically 200 nm, making SPR a surface-sensitive technique ideal forstudying interactions between immobilized biomolecules and asolution-phase analyte. SPR technology offers several advantages overconventional techniques, such as fluorescence or ELISA (enzyme-linkedimmunosorbent assay) based approaches. First, because SPR measurementsare based on refractive index changes, detection of an analyte is labelfree and direct. The analyte does not require any specialcharacteristics or labels (radioactive or fluorescent) and can bedetected directly, without the need for multistep detection protocols.Secondly, the measurements can be performed in real time, allowing theuser to collect kinetic data, as well as thermodynamic data. Lastly, SPRis a versatile technique, capable of detecting analytes over a widerange of molecular weights and binding affinities. Therefore, SPRtechnology is a powerful tool for studying biomolecule interactions. Sofar, in research settings, SPR based techniques have been used toinvestigate protein—peptide interactions, cellular ligation, protein—DNAinteractions, and DNA hybridization. However, SPR based approaches havenot yet been explored in clinical medicine, especially in clinicallaboratory medicine.

The present invention relates to the application of SPR technology inmedical diagnostics, i.e., the detection of human papillomavirus (HPV)genotypes. HPV is one of the most common causes of sexually transmitteddiseases. At least 200 types of HPV have been identified, and 85 of themhave been well characterized to date. Cervical cancer is one of theglobally most common cancers in women. The correlation between genitalHPV infections and cervical cancer was first reported in the early1980s. Genital HPV infections have been implicated in 99.7% of allcervical squamous cell carcinomas. More than 35 different HPV genotypeshave been associated with cervical epithelial neoplasia (CIN) andcervical cancer. According to their association with cervical cancer andprecursor lesions, HPVs are divided into two groups: high-risk andlow-risk HPV. The high-risk group includes types 16, 18, 31, 33, 35, 39,45, 51, 52, 53, 56, 58, 59, 66, 68 and the low-risk group types 6, 11,34, 40, 42, 43, 44, 54, 70. Traditional methods for HPV detection, ingeneral, have showed low sensitivity and specificity. Moreover, theseapproaches are either very labor intensive or requiring fluorescentlabels, and can not detect specific HPV types simultaneously. SPRtechnology has the ability of providing unlabel, high-throughput, andon-line parallel analysis, and has been demonstrated by us to serve as apowerful tool in detecting HPV genotypes.

REFERENCES

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MODES FOR CARRYING OUT THE INVENTION

Before the present method of using SPR technology to detect humanpapillomavirus (HPV) genotypes is disclosed and described, it is to beunderstood that this invention is not limited to the particularconfigurations, process steps, and materials disclosed herein as suchconfigurations, process steps, and materials may vary somewhat. It isalso to be understood that the terminology employed herein is used forthe purpose of describing particular embodiments only and is notintended to be limiting since the scope of the present invention will belimited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference “a HPV probe” includes reference to two or more such HPVprobes.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions set outbelow.

As used herein, a “metal binding tag” refers to a group of moleculesthat can become fastened to a metal that is coordinated by a chelate.Suitable groups of such molecules include amino acid sequencesincluding, but not limited to, histidines and cysteines (“polyamino acidtags”). Metal binding tags include histidine tags, defined below.

“Signaling entity” means an entity that is capable of indicating itsexistence in a particular sample or at a particular location. Signalingentities of the invention can be those that are identifiable by theunaided human eye, those that may be invisible in isolation but may bedetectable by the unaided human eye if in sufficient quantity (e.g.,colloid particles), entities that absorb or emit electromagneticradiation at a level or within a wavelength range such that they can bereadily determined visibly (unaided or with a microscope including anelectron microscope or the like), or spectroscopically, entities thatcan be determined electronically or electrochemically, such asredox-active molecules exhibiting a characteristic oxidation/reductionpattern upon exposure to appropriate activation energy (“electronicsignaling entities”), or the like. Examples include dyes, pigments,electroactive molecules such as redox-active molecules, fluorescentmoieties (including, by definition, phosphorescent moieties),up-regulating phosphors, chemiluminescent entities,electrochemiluminescent entities, or enzyme-linked signaling moietiesincluding horse radish peroxidase and alkaline phosphatase.

“Precursors of signaling entities” are entities that by themselves maynot have signaling capability but, upon chemical, electrochemical,electrical, magnetic, or physical interaction with another species,become signaling entities. An example includes a chromophore having theability to emit radiation within a particular, detectable wavelengthonly upon chemical interaction with another molecule. Precursors ofsignaling entities are distinguishable from, but are included within thedefinition of, “signaling entities” as used herein.

As used herein, “fastened to or adapted to be fastened”, in the contextof a species relative to another species or to a surface of an article,means that the species is chemically or biochemically linked viacovalent attachment, attachment via specific biological binding (e.g.,biotin/streptavidin), coordinative bonding such as chelate/metalbinding, or the like. For example, “fastened” in this context includesmultiple chemical linkages, multiple chemical/biological linkages, etc.,including, but not limited to, a binding species such as a peptidesynthesized on a polystyrene bead, a binding species specificallybiologically coupled to an antibody which is bound to a protein such asprotein A, which is covalently attached to a bead, a binding speciesthat forms a part (via genetic engineering) of a molecule such as GST orPhage, which in turn is specifically biologically bound to a bindingpartner covalently fastened to a surface (e.g., glutathione in the caseof GST), etc. As another example, a moiety covalently linked to a thiolis adapted to be fastened to a gold surface since thiols bind goldcovalently. Similarly, a species carrying a metal binding tag is adaptedto be fastened to a surface that carries a molecule covalently attachedto the surface (such as thiol/gold binding) and which molecule alsopresents a chelate coordinating a metal. A species also is adapted to befastened to a surface if that surface carries a particular nucleotidesequence, and the species includes a complementary nucleotide sequence.

“Covalently fastened” means fastened via nothing other than by one ormore covalent bonds, e.g. a species that is covalently coupled, viaEDC/NHS chemistry, to a carboxylate-presenting alkyl thiol which is inturn fastened to a gold surface, is covalently fastened to that surface.

“Specifically fastened (or bound)” or “adapted to be specificallyfastened (or bound)” means a species is chemically or biochemicallylinked to another specimen or to a surface as described above withrespect to the definition of “fastened to or adapted to be fastened”,but excluding all non-specific binding.

“Non-specific binding”, as used herein, is given its ordinary meaning inthe field of biochemistry.

As used herein, a component that is “immobilized relative to” anothercomponent either is fastened to the other component or is indirectlyfastened to the other component, e.g., by being fastened to a thirdcomponent to which the other component also is fastened, or otherwise istranslationally associated with the other component. For example, asignaling entity is immobilized with respect to a binding species if thesignaling entity is fastened to the binding species, is fastened to acolloid particle to which the binding species is fastened, is fastenedto a dendrimer or polymer to which the binding species is fastened, etc.A colloid particle is immobilized relative to another colloid particleif a species fastened to the surface of the first colloid particleattaches to an entity, and a species on the surface of the secondcolloid particle attaches to the same entity, where the entity can be asingle entity, a complex entity of multiple species, a cell, anotherparticle, etc.

The term “sample” refers to any medium suspected of containing ananalyte, such as a binding partner, the presence or quantity of which isdesirably determined. The sample can be a biological sample such as acell, cell lysate, tissue, serum, blood or other fluid from a biologicalsource, a biochemical sample such as products from a cDNA library, anenvironmental sample such as a soil extract, or any other medium,biological or non-biological, including synthetic material, that canadvantageously be evaluated in accordance with the invention.

A “sample suspected of containing” a particular component means a samplewith respect to which the content of the component is unknown. Thesample may be unknown to contain the particular component, or may beknown to contain the particular component but in an unknown quantity.

As used herein, a “metal binding tag” refers to a group of moleculesthat can become fastened to a metal that is coordinated by a chelate.Suitable groups of such molecules include amino acid sequences,typically from about 2 to about 10 amino acid residues. These include,but are not limited to, histidines and cysteines (“polyamino acidtags”). Such binding tags, when they include histidine, can be referredto as a “poly-histidine tract” or “histidine tag” or “HIS-tag”, and canbe present at either the amino- or carboxy-terminus, or at any exposedregion of a peptide or protein or nucleic acid. A poly-histidine tractof six to ten residues is preferred for use in the invention. Thepoly-histidine tract is also defined functionally as being the number ofconsecutive histidine residues added to a protein of interest whichallows for the affinity purification of the resulting protein on a metalchelate column, or the identification of a protein terminus throughinteraction with another molecule (e.g. an antibody reactive with theHIS-tag).

A “moiety that can coordinate a metal”, as used herein, means anymolecule that can occupy at least two coordination sites on a metalatom, such as a metal binding tag or a chelate.

“Affinity tag” is given its ordinary meaning in the art. Affinity tagsinclude, for example, metal binding tags, GST (in GST/glutathionebinding clip), and streptavidin (in biotin/streptavidin binding). Atvarious locations herein specific affinity tags are described inconnection with binding interactions. It is to be understood that theinvention involves, in any embodiment employing an affinity tag, aseries of individual embodiments each involving selection of any of theaffinity tags described herein.

The term “self-assembled monolayer” (SAM) refers to a relatively orderedassembly of molecules spontaneously chemisorbed on a surface, in whichthe molecules are oriented approximately parallel to each other androughly perpendicular to the surface. Each of the molecules includes afunctional group that adheres to the surface, and a portion thatinteracts with neighboring molecules in the monolayer to form therelatively ordered array. See Laibinis. P. E.; Hickman. J.: Wrighton. M.S.: Whitesides, G. M. Science 245, 845 (1989). Bain. C.; Evall. J.:Whitesides. G. M. J. Am. Chem. Soc. 111, 7155-7164 (1989), Bain, C.;Whitesides, G. M. J. Am. Chem. Soc. 111, 7164-7175 (1989), each of whichis incorporated herein by reference. The SAM can be made up completelyof SAM-forming species that form close-packed SAMs at surfaces, or thesespecies in combination with molecular wires or other species able topromote electronic communication through the SAM (includingdefect-promoting species able to participate in a SAM), or other speciesable to participate in a SAM, and any combination of these. Preferably,all of the species that participate in the SAM include a functionalitythat binds, optionally covalently, to the surface, such as a thiol whichwill bind covalently to a gold surface. A self-assembled monolayer on asurface, in accordance with the invention, can be comprised of a mixtureof species (e.g. thiol species when gold is the surface) that canpresent (expose) essentially any chemical or biological functionality.For example, they can include tri-ethylene glycol-terminated species(e.g. tri-ethylene glycol-terminated thiols) to resist non-specificadsorption, and other species (e.g. thiols) terminating in a bindingpartner of an affinity tag, e.g. terminating in a chelate that cancoordinate a metal such as nitrilotriacetic acid which, when in complexwith nickel atoms, captures a metal binding tagged-species such as ahistidine-tagged binding species.

“Molecular wires” as used herein, means wires that enhance the abilityof a fluid encountering a SAM-coated electrode to communicateelectrically with the electrode. This includes conductive molecules or,as mentioned above and exemplified more fully below, molecules that cancause defects in the SAM allowing communication with the electrode. Anon-limiting list of additional molecular wires includes2-mercaptopyridine, 2-mercaptobenzothiazole, dithiothreitol,1,2-benzenedithiol, 1,2-benzenedimethanethiol, benzene-ethanethiol, and2-mercaptoethylether. Conductivity of a monolayer can also be enhancedby the addition of molecules that promote conductivity in the plane ofthe electrode. Conducting SAMs can be composed of, but are not limitedto: 1) poly (ethynylphenyl) chains terminated with a sulfur; 2) an alkylthiol terminated with a benzene ring; 3) an alkyl thiol terminated witha DNA base; 4) any sulfur terminated species that packs poorly into amonolayer; 5) all of the above plus or minus alkyl thiol spacermolecules terminated with either ethylene glycol units or methyl groupsto inhibit non specific adsorption. Thiols are described because oftheir affinity for gold in ready formation of a SAM. Other molecules canbe substituted for thiols as known in the art from U.S. Pat. No.5,620,820, and other references. Molecular wires typically, because oftheir bulk or other conformation, create defects in an otherwiserelatively tightly-packed SAM to prevent the SAM from tightly sealingthe surface against fluids to which it is exposed. The molecular wirecauses disruption of the tightly-packed self-assembled structure,thereby defining defects that allow fluid to which the surface isexposed to communicate electrically with the surface. In this context,the fluid communicates electrically with the surface by contacting thesurface or coming in close enough proximity to the surface thatelectronic communication via tunneling or the like can occur.

The term “biological binding” refers to the interaction between acorresponding pair of molecules that exhibit mutual affinity or bindingcapacity, typically specific or non-specific binding or interaction,including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, antibody/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

The term “binding” or “bound” refers to the interaction between acorresponding pair of molecules that exhibit mutual affinity or bindingcapacity, typically specific or non-specific binding or interaction,including biochemical, physiological, and/or pharmaceuticalinteractions. Biological binding defines a type of interaction thatoccurs between pairs of molecules including proteins, nucleic acids,glycoproteins, carbohydrates, hormones and the like. Specific examplesinclude antibody/antigen, anti body/hapten, enzyme/substrate,enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrierprotein/substrate, lectin/carbohydrate, receptor/hormone,receptor/effector, complementary strands of nucleic acid,protein/nucleic acid repressor/inducer, ligand/cell surface receptor,virus/ligand, etc.

The term “binding partner” refers to a molecule that can undergo bindingwith a particular molecule. Biological binding partners are examples.For example, Protein A is a binding partner of the biological moleculeIgG, and vice versa.

The term “determining” refers to quantitative or qualitative analysis ofa species via, for example, spectroscopy, ellipsometry, piezoelectricmeasurement, immunoassay, electrochemical measurement, and the like.“Determining” also means detecting or quantifying interaction betweenspecies, e.g. detection of binding between two species.

The term “self-assembled mixed monolayer” refers to a heterogeneousself-assembled monolayer, that is, one made up of a relatively orderedassembly of at least two different molecules.

“Synthetic molecule”, means a molecule that is not naturally occurring,rather, one synthesized under the direction of human or human-created orhuman-directed control.

The present invention generally relates to a method of using SPRtechnology to detect human papillomavirus (HPV) genotypes. In addition,the present invention provides an efficient formula to make a mixed SAMthat can greatly enhance the immobilization ability of the metalsurface, which is desirable for the immobilization of relevant HPVprobes for detection. The HPV probes can be selected specific probes forthe high risk types 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59,66, 68 and the low-risk group types 6, 11, 34, 40, 42, 43, 44, 54, 70.

To enhance the sensitivity and specificity of the SPR technology, alinking layer is attached onto the gold film on the surface of a glasschip that serves as a functional structure for further modification ofthe gold film surface. So far, several immobilization chemistries aresuitable for the formation of the linking layer, including alkanethiols,hydrogel, silanes, polymer films and polypeptides. Moreover, there areseveral methods to attach the linking layer onto the thin gold surface,such as the Langmuir-Blodgett film method and the self-assembledmonolayer (SAM) approach.

The following examples will enable those skilled in the art to moreclearly understand how to practice the present invention. It is to beunderstood that, while the invention has been described in conjunctionwith the preferred specific embodiments thereof, that which follows isintended to illustrate and not limit the scope of the invention. Otheraspects of the invention will be apparent to those skilled in the art towhich the invention pertains.

Example 1 Detection of HPV Genotypes from the Vaginal Swabs from Cervix

Testing Sample: the Vaginal Swab from Cervix.

HPV probes represented: specific probes for the high risk types 16, 18,31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and the low-riskgroup types 6, 11, 34, 40, 42, 43, 44, 54, 70. Oligonucleotide probes at20-60 nt were synthesized and labeled at 5′ terminal with —SH or biotinso that the probes could form a monolayer on the bare gold surface orbind on the modified chip surface.

Formation of a link layer on the surface of a gold-film glass chip

In order to enhance the analytic sensitivity and specificity of SPRtechnology a link layer is attached onto the gold film on the surface ofa glass chip to serve as a functional structure for further modificationof the gold film surface. So far, several immobilization chemistries aresuitable for the formation of the link layer, including alkanethiols,hydrogel, silanes, polymer films and polypeptides. Moreover, there areseveral methods to attach the link layer onto the thin gold surface,such as Langmuir-Blodgett film method and self-assembled monolayer (SAM)approach.

In this example, alkanethiols are chosen to form a mixed SAM on thesurface of a gold film because a mixed SAM of long-chain alkanethiolscan bind with biomolecules through their suitable reactive groups (suchas carboxyl-terminal) on one side and react with the gold film through agold-complexing thiol on the other side. In detail, ten millimolar mixedsolutions consisting of 10:1 molar ratios of 3-mercaptopropanol (3-MPOH)to 11-mercaptoundecanoic acid (11-MUA) are prepared in pure ethanol. Theprepared gold films are immersed in the solutions for 24 h and then arerinsed several times with ethanol and deionized water. After rinsing,the gold films are dried in a pure N₂ gas stream.

By comparing different alkanethiols, an efficient formula is generated,i.e., ten millimolar mixed solutions consisting of 10:1 molar ratios of3-mercaptopropanol (3-MPOH) to 11-mercaptoundecanoic acid (11-MUA), fromwhich to make a mixed SAM that is good for the immobilization ofrelevant HPV probes.

Immobilization of HPV probes on the surface of the link layer

Probes modified with —SH can form a monolayer on the gold film. Probeslabeled with biotin can bind on the streptavidin (SA) chip; the probesmodified with biotin are bound on the chip by amine coupling method.

To improve the orientation of the captured biomolecules and to reducenon-specific binding, the biotin-streptavidin system is employed in thisinvention. First, either the carboxyl-terminated groups of a SAM arebiotinylated followed with subsequent binding of streptavidin, orstreptavidin is directly immobilized to the SAM, depending on themolecular weight of detected molecules. In detail, the flow rates of allsolutions are maintained at 5 μl/min during immobilization. Thelink-layer/gold-film glass chips as prepared above are rinsed in 0.1MMES buffer (pH 4.7-5.5). Afterwards, they are soaked in a clean bottlecontaining 5 ml of 0.1M MES buffer (2-morpholinoethane sulfonic acid)with the gold-coated layers facing upward. The carboxyl groups areactivated by adding 65 μl of 100 mg/mlEDC(N-ethyl-N_-(3-diethylaminopropyl) carbodiimide), and then conjugatedwith 130 μl of biotin hydrazide (50 mM). After 12 h at room temperaturewith gentle shaking, the chips are cleaned several times with ultrapurewater and HBS buffer (pH 7.0). Finally, the chips are cleaned and driedunder a pure N₂ gas stream. Then streptavidin is immobilized byinjecting streptavidin (20 μg/ml in HBS buffer pH 7.4) for 7 min.

To immobilize streptavidin directly to a SAM, the SAM surface is firstequilibrated with HBS buffer for about 30 min to obtain a stablebaseline. After obtaining a stable baseline, terminal carboxylic groupsof the mixed SAM are activated with a 7 min pulse of a 1:1 mixture of0.1M NHS and 0.1M EDC, and then streptavidin (200 μg/ml) in 10 mM sodiumacetate buffer at pH 5.5 is injected for 15 min. After immobilization ofthe streptavidin, 1.0 Methanolamine-HCl is flowed over the SAM surfacefor 10 min to block the remaining active sites, which is also effectivefor blocking non-specific binding. Secondly, the HPV probes representedare biotinylated by technique according to the standard protocol.

Measuring the level of biotin incorporation is carried out with anEZ-link sulfo-NHS-LCBiotinylation Kit according to the manufacturer'sprotocol. Afterwards, the biotinylated HPV probes are denatured at 98°C. for about 5 min, and then quickly cooled in ice to make the markersbeing single stranded. Lastly, the single-strand and biotinylated HPVprobes covalently bind to the streptavidin attached to the SAM. Briefly,the biotinylated HPV probes each at about 1-2 ng/ul in TE buffer areinjected into each array cell, respectively, for 7 min. The unboundbiotinylated HPV probes are washed away by using a mixed solution of 25mM NaOH/0.2M NaCl for 2 min.

Immobilization of thiol-derivative probes on the bare gold sensor chip

The gold sensor chip was cleaned with a solution consisting of H₂O₂(30%), NH3 (30%) and milliQ water in a 1:1:5 ratio for 10 min and thenthoroughly washed with milliQ water. After the cleaning step, the sensorchip was covered with a solution (1 uM, 1 ml) of thiolated probe inimmobilization solution (KH₂PO₄ 1M, pH 3.8.) and incubated at roomtemperature for 2 h. Following the incubation, the sensor chip waswashed with milliQ water and treated with 1 mM (1 ml) of blocking thiolsolution (MCH, 1 uM) at room temperature for one hour in the dark. Afterwashing with water, the chip was left to dry to be mounted onto theplastic support and docked into the SPR instrument ready forhybridization reactions.

Testing a Sample

Based on the standard protocol, vaginal swab from the cervix wascollected and DNA was extracted with an appropriate method or acommercially available DNA isolation kit.

To obtain enough HPV DNA, L1 regions of HPV DNA were amplified usingPCR. In this invention, asymmetric PCR was used to obtain single strandDNA (ssDNA). The PCR amplification system was offered in a reaction mixform, containing 10 mM Tris-HCL at pH8.3, 50 mM KCL, 1.0-5.0 mM MgCL2,100-500 uM deoxynucleotide triphosphates (dNTP) each, 0.5-4 u/ul DNApolymerase, 0.01-5.0 uM primers each and any other elements necessaryfor DNA amplification except DNA template. A normal control sample wasperformed simultaneously as well.

Herein modified consensus HPV GP5d+/GP6d+ primers were used to amplifyall HPV genotypes. An oligonucleotide linker (between 10 to 30nucleotides) with no targets on other sites of HPV genomes was linked tothe 5′ end of the sense or antisense primer. The link can improve theprimer's temperature for more than 10° C. The concentration ratiobetween the primers with and without the linker should range from 0.01to 1.

The kits can also be offered in a separate form with primers, DNApolymerase, buffer and dNTP apart from each other.

The reaction mix can be dispensed into 20-50 ul volumes. 10 ng-1 ug ofhuman genome DNA containing HPV DNA in a volume of about 1.0-5 ul shouldbe added into each portion. The thermal cycler can be set up as follows:

3 to 5 minutes at 94° C. for 1 cycle; 15 seconds to 1 minute at 94° C.,30 seconds to 1 minute at 45-60° C., 15 seconds to 1 minute at 72° C.for 15 to 30 cycles; 15 seconds to 1 minute at 94° C., 30 seconds to 1minute at 60-70° C., 15 seconds to 1 minute at 72° C. for 25 to 50cycles; 0-10 min at 72° C. for 1 cycle.

On completion of the amplification program, samples may be stored atroom temperature overnight or at 2-8° C. for up to 7 days beforeanalysis.

Hybridization experiments were conducted with a SPR instrument at 37° C.with a flow rate of 5 ul/min injecting 100 ul of sample solution ontothe probe-immobilized chip. Amplified HPV DNA was diluted with thehybridization solution for 1 to 10 times. The hybridization solutioncontained 3×SSPE, 5×Denhardt's, 0.1×TE(pH8.0) and 0.5% SDS. The reactionwas monitored for 20-40 min and then the sensor chip was automaticallywashed with hybridization buffer to remove the unbound DNA material. Theanalytical signal, reported as resonance units)(m°), was derived fromthe different values between the HPV specific probes and the negativecontrol probe(s), which is called on-line hybridization method.

The hybridization experiments can also be conducted off a SPRinstrument, which is called off-line hybridization method. Theadvantages of off-line hybridization method are that the temperature andtime for hybridization can be controlled easily.

With observation of the difference between a specific HPV probe and anegative control probe greater than 50 m° [what is this mean?]hybridization value, it would indicate the presence of the specific HPVgenotype in a sample.

The following is a more detailed description of the procedure for chippreparation and probe preparation: the probes can be synthesized and belabeled by —SH or biotin so that the probes can form monolayer on thebare gold chip surface or bind on the modified chip surface. The probesshould be denatured to single stands prior to use. If oligonucleotideprobes (20-60 bp) are used, the probes can be synthesized and the —SH orbiotin can be added to the probe terminus. Once the probes are denaturedto single strands, the probes can be immobilizes on the chip surface.

Immobilization of thiol-labeled probes on the bare gold chip surface:the gold sensor chip was cleaned with a solution consisting of H₂O₂(30%), NH₃ (30%) and milliQ water in a 1:1:5 ratio for 10 min and thenthoroughly washed with milliQ water. After the cleaning step, the sensorchip was covered with a solution (1 uM, 1 ml) of thiolated probes inimmobilization solution (KH₂PO₄ 1M, pH 3.8.) and incubated at roomtemperature for 2 h. Afterwards, the sensor chip was washed with milliQwater and treated with 1 mM (1 ml) blocking thiol solution (MCH, 1 uM)at room temperature for one hour in dark. After washing with water, itwas left to dry to be mounted onto the plastic support and docked intothe SPR instrument ready for hybridization reactions.

Immobilization of oligonucleotide probes labeled with biotin on themodified chip surface: immobilization of the oligonucleotide probeslabeled with biotin can use the streptavidin—biotin method.

The dextran-modified chips were made with the following description:

Cleanliness of Substrate

Metal substrates (copper, silver, aluminum or gold) were cleaned withstrong oxidizing chemicals (“piranha” solution-H₂SO₄:H₂O₂) or argonplasmas, and their surfaces were washed with ultra pure water anddegassed ethanol. After rinsing, the substrates were dried with pure N₂gas stream.

Preparation of Self-Assembled Monolayers

Single-component or mixed self-assembled monolayers (SAMs) oforganosulfur compounds (thiols, disulfides, sulfides) on the clean metalsubstrate have been widely applied for chemical modification to developchemical and biological sensor chips. Preparing SAMs on metal substrateswas achieved by immersion of a clean substrate into a dilute (˜1-10 m M)ethanolic solution of organosulfur compounds for 12-18 h at roomtemperature.

Monolayers comprising a well-defined mixture of molecular structures arecalled “mixed” SAMs. There are three easy methods for synthesizing mixedSAMs: (1) coadsorption from solutions containing mixtures ofalkanethiols (HS(CH₂)_(n)R+HS(CH₂)_(n)R′), (2) adsorption of asymmetricdialkyl disulfides (R(CH₂)_(m)S—S(CH₂)_(n)R′), and (3) adsorption ofasymmetric dialkylsulfides (R(CH₂)_(m)S(CH₂)_(n)R′), where n and m arethe number of methylene units (range from 3 to 21) and R represents theend group of the alkyl chain (—CH₃, —OH, —COOH, NH₂) active forcovalently binding ligands or biocompatible substance. Mixed SAMs areuseful for decreasing the steric hindrance of interfacial reaction that,in turn, is useful for studying the properties and biology of cells.

Rather than using single-component for preparing the SAM in conventionalmethods, “mixed” SAMs were used in the present invention, which providesvarious functional groups and branching structures to decrease thesteric hindrance of interfacial reaction that, in turn, is useful forstudying the biomolecular interaction analysis.

Methods for modifying SAMs after their formation are critical for thedevelopment of surfaces that present the large, complex ligands andmolecules needed for biology and biochemistry. There are two importanttechniques for modifying SAMs:

(1) Direct reaction with exposed functional groups: under appropriatereaction conditions, terminal functional groups (—OH, —COOH) exposed onthe surface of a SAM immersed in a solution of ligands could reactdirectly with the molecules present in solution. Many directimmobilization techniques have been adapted from methods forimmobilizing DNA, polypeptides, and proteins on SAMs

(2) Activation of surfaces for reactions: an operationally differentapproach to the functionalization of the surfaces of SAMs is to form areactive intermediate, which is then coupled to a ligand. In thisinvention, we chose epoxy activation method to couple polysaccharide ora swellable organic polymer. In detail, 2-(2-Aminoethoxy)ethanol (AEE)was coupled to carboxyl-functionalized SAM using peptide couplingreagents(N-hydroxysuccinimide/N-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide(EDC/NHS)), and the terminal hydroxyl groups were further reacted withepichlorohydrin to produce epoxy-functionalized surfaces. These weresubsequently reacted with hydroxyl moieties of polysaccharide or organicpolymer. Subsequently, the polysaccharide chains were carboxylatedthrough treatment with bromoacetic acid more than one time. Theresultant material offered for further functionalization withbiomolecules.

Streptavidin Immobilized on the Dextran-Modified Chip Surface

35 ul of a solution containing 50 mM NHS and 200 mM EDAC in water wereinjected to activate the dextran-modified surface. The chip was furthermodified with streptavidin (200 ug/ml in acetate buffer 10 mM, pH5.0).Then, the residual reacting sites were blocked with 35 ul solution ofethanolamine hydrochloride (pH 8.6, 1M water solution). Finally, thebiotinylated predenatured probe was added (100 ul probe, 1 uM inimmobilization buffer (NaCl 300 mM, Na₂HPO₄ 20 mM, EDTA 0.1 mM, pH 7.4).

Sample DNA Preparation

The DNA can be extracted by using commercial extraction kits. Ifnecessary, the DNA can be further amplified by using methods, such asconventional PCR, RT-PCR, nested-PCR, DOP-PCR, random-PCR, etc.

Sample DNA Denaturing and Blocking

Prior to SPR testing, sample DNA needs to be pre-treated to becomesingle-stranded available for hybridization to the immobilized probe. Ifneeded, the DNA may be treated by supersonic or endonuclease.

The high temperature denaturing method was employed. This method wasfound to be a simple and useful way to obtain single-stranded DNAavailable for hybridization. The principle of this method relies on theuse of small (20 bases) oligonucleotides added to the denaturationmixture. These oligonucleotides are complementary to some sequences onthe strand that hybridizes to the immobilized probe. By the interactionbetween the thermally separated DNA strands and these oligonucleotides,surface hybridization can occur. The whole denaturation procedure wascombined with sense and antisense primers. The protocol was composed bya 5 min incubation step at 95° C. and then 1 min at 50° C., suitable forprimers annealing in the PCR procedure. Cot1 DNA, salmon sperm DNA oryeast tRNA etc. were added into the denaturation system to block thechip so that the background and the nonspecific hybridization could beminimized.

Hybridization

Hybridization experiments were conducted in the SPR instrument at a flowrate of 5 ul/min (at 25° C.) injecting 25 ul of the sample DNA asblocked by cot I DNA on the probe immobilized chip. The reaction wasmonitored for 5 min and then the sensor chip was automatically washedwith hybridization buffer to remove the unbound DNA material. Theanalytical signal, reported as resonance units (RU), was derived fromthe difference between the final value and the value recorded before thetarget injection (baseline). It is referred as on-line hybridizationmethod. The hybridization experiments can also be conducted off the SPRinstrument, which is referred as off-line hybridization method.Advantage of the off-line hybridization method is that temperature andtime can be controlled easily for the experiments.

It is to be understood that the above-described embodiments are onlyillustrative of application of the principles of the present invention.Numerous modifications and alternative embodiments can be derivedwithout departing from the spirit and scope of the present invention andthe appended claims are intended to cover such modifications andarrangements. Thus, while the present invention has been shown in thedrawings and fully described above with particularity and detail inconnection with what is presently deemed to be the most practical andpreferred embodiment(s) of the invention, it will be apparent to thoseof ordinary skill in the art that numerous modifications can be madewithout departing from the principles and concepts of the invention asset forth in the claims.

1. An improved SPR biosensor chip for simultaneous detection of multipleHPV genotypes prepared by forming a linking layer on the surface of ametal film on a glass chip and immobilizing of multiple HPV probes onthe surface of the linking layer.
 2. The improved SPR biosensor chipaccording to claim 1, wherein the linking layer is prepared by preparinga mixed SAM of long-chain alkanethiols which can bind with biomoleculesthrough its suitable reactive groups on one side and react with saidgold film through a gold-complexing thiol on the other side, modifyingand activating the mixed SAMs.
 3. The improved SPR biosensor chipaccording to claim 1, wherein said metal film is treated with dextranusing 2-(2-Aminoethoxy)ethanol (AEE) as a crosslinking agent andmultiple bromoacetic acid reactions.
 4. The improved SPR biosensor chipaccording to claim 2, wherein said mixed SAMs are prepared by one of thefollowing: (1) coadsorption from solutions containing mixtures ofalkanethiols (HS(CH₂)_(n)R+HS(CH₂)_(n)R′), (2) adsorption of asymmetricdiallyl disulfides (R(CH₂)_(m)S—S(CH₂)_(n)R′), and (3) adsorption ofasymmetric dialkylsulfides (R(CH₂)_(m)S(CH₂)_(n)R′), wherein n and m arethe number of methylene units which is an integer from 3 to 21 and Rrepresents the end group of the alkyl chain (—CH₃, —OH, —COOH, NH₂)active for covalently binding ligands or biocompatible substance.
 5. Theimproved SPR biosensor chip according to claim 2, wherein said modifyingand activating the mixed SAMs is accomplished by an epoxy activationmethod to couple a polysaccharide or a swellable organic polymercomprising coupling 2-(2-Aminoethoxy)ethanol (AEE) tocarboxyl-functionalized SAM using peptide coupling reagents(N-hydroxysuccinimide/N-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide(EDC/NHS)), and reacting with epichlorohydrin to produceepoxy-functionalized surfaces, which subsequently being reacted withhydroxyl moieties of the polysaccharide or organic polymer, theresulting polysaccharide chains are subsequently being carboxylatedthrough treatment with bromoacetic acid multiple times.
 6. The improvedSPR biosensor chip according to claim 1, wherein said HPV probes areselected from the group consisting of specific probes for the high risktypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and t.The low-risk group included types 6, 11, 34, 40, 42, 43, 44, 54,
 70. 7.The improved SPR biosensor chip according to claim 1, wherein said HPVprobes are immobilized to the surface of the linking layer using abiotin-streptavidin system or —SH as the immobilization agent.
 8. Theimproved SPR biosensor chip according to claim 1, wherein said metal iscopper, silver, aluminum or gold.
 9. A method for simultaneouslydetecting multiple HPV genotypes in a cervical sample, comprising thesteps of: 1) preparing a surface plasmon resonance (SPR) systemcomprising: a) an improved SPR biosensor chip according to claim 1; b) aspectrophotometric means for receiving a first signal and a secondsignal from said biosensor surface, said second signal being received ata time after hybridization reaction of the sample to be tested and saidHPV probes on said biosensor surface; and c) means for calculating andcomparing properties of said first received signal and said secondreceived signal to determine the presence of said HPV genotypes; 2)preparing a DNA extract from a cervical sample to be tested andutilizing asymmetric PCR method and modified consensus HPV GP5d+/GP6d+primers to amplify ssDNA, contacting the resulting amplified DNApreparation with said biosensor and spectrophotometrically receivingsaid first signal and said second signal; 3) calculating the differencesbetween said received first and second signals.
 10. The improved SPRbiosensor chip according to claim 9, wherein the linking layer isprepared by preparing a mixed SAM of long-chain alkanethiols which canbind with biomolecules through its suitable reactive groups on one sideand react with said gold film through a gold-complexing thiol on theother side, modifying and activating the mixed SAMs.
 11. The improvedSPR biosensor chip according to claim 9, wherein said metal film istreated with dextran using 2-(2-Aminoethoxy)ethanol (AEE) as acrosslinking agent and multiple bromoacetic acid reactions.
 12. Theimproved SPR biosensor chip according to claim 10, wherein said mixedSAMs are prepared by one of the following: (1) coadsorption fromsolutions containing mixtures of alkanethiols(HS(CH₂)_(n)R+HS(CH₂)_(n)R′), (2) adsorption of asymmetric dialkyldisulfides (R(CH₂)_(m)S—S(CH₂)_(n)R′) and (3) adsorption of asymmetricdialkylsulfides (R(CH₂)_(m)S(CH₂)_(n)R′), wherein n and m are the numberof methylene units which is an integer from 3 to 21 and R represents theend group of the alkyl chain (—CH₃, —OH, —COOH, NH₂) active forcovalently binding ligands or biocompatible substance.
 13. The improvedSPR biosensor chip according to claim 10, wherein said modifying andactivating the mixed SAMs is accomplished by an epoxy activation methodto couple a polysaccharide or a swellable organic polymer comprisingcoupling 2-(2-Aminoethoxy)ethanol (AEE) to carboxyl-functionalized SAMusing peptide coupling reagents(N-hydroxysuccinimide/N-Ethyl-N′-(3-dimethylaminopropyl)-carbodiimide(EDC/NHS)), and reacting with epichlorohydrin to produceepoxy-functionalized surfaces, which subsequently being reacted withhydroxyl moieties of the polysaccharide or organic polymer, theresulting polysaccharide chains are subsequently being carboxylatedthrough treatment with bromoacetic acid multiple times.
 14. The improvedSPR biosensor chip according to claim 9, wherein said HPV probes areselected from the group consisting of specific probes for the high risktypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68 and t.The low-risk group included types 6, 11, 34, 40, 42, 43, 44, 54,
 70. 15.The improved SPR biosensor chip according to claim 9, wherein said HPVprobes are immobilized to the surface of the linking layer using abiotin-streptavidin system or —SH as the immobilization agent.
 16. Theimproved SPR biosensor chip according to claim 9, wherein said metal iscopper, silver, aluminum or gold.